This invention relates generally to medical treatment methods. Specifically, the invention relates to methodology for the correction of defective chloride transport by activation of chloride channels of the lung and other epithelia using genetic or chemical modification. These methods relate to the treatment of epithelia with compounds which cause activation of the channel as measured by increased probability (Po) of opening of the channel at physiologically relevant holding potentials. These methods also relate to the treatment of epithelia with gene therapy to introduce chloride channels genes with site mutations which cause activation of the channel as measured by increased probability (Po) of opening of the channel at physiologically relevant holding potentials. These treatments will reduce life-threatening complications frequently found in diseases such as cystic fibrosis. These methods of activation of chloride channels also comprise treatment of chloride channels with amidation reactions.
Cystic fibrosis is a lethal disease affecting approximately one in 2,500 live Caucasian births and is the most common autosomal recessive disease in Caucasians. Patients with this disease have reduce chloride ion permeability in the secretory and absorptive cells of organs with epithelial cell linings, including the airways, pancreas, intestine, sweat glands and male genital tract. This, in turn, reduces the transport of water across the epithelia. The lungs and the GI tract are the predominant organ systems affected in this disease and the pathology is characterized by blocking of the respiratory and GI tracts with viscous mucus. The chloride impermeability in affected tissues is due to mutations in a specific chloride channel, the cystic fibrosis transmembrane conductance regulator protein (CFTR), which prevents normal passage of chloride ions through the cell membrane (Welsh et al., Neuron, 8:821-829 (1992)). There is no effective treatment for the disease, and therapeutic research is focused on gene therapy and/or activating the defective or other chloride channels in the cell membrane to normalize chloride permeability (Tizzano et al., J. Pediat., 120:337-349 (1992)). Damage to the lungs due to mucus blockage, frequent bacterial infections and inflammation is the primary cause of morbidity and mortality in CF patients and, although maintenance therapy has improved the quality of patients' lives, the median age at death is still only around 30 years.
The thick build-up of mucus deposits in the lungs leads to a higher than normal susceptibility towards fatal pulmonary infections. It is these infections, often of the Pseudomonas aeruginosa type, that are generally the causative agents of cystic fibrosis related death. At present, the established treatment protocols for cystic fibrosis involve treating these secondary infections with appropriate antibiotics, as well as adjusting diet and removing by physical means the deleterious build up of mucociliary secretions. Thus considerable current effort is being devoted to developing treatments that operate by attacking the underlying cause of disease. Here, a variety of approaches have been explored. These range from attempts at gene therapy (incorporating the normal, wild-type cystic fibrosis gene into epithelia cells) to the administration of agents that restore electrolyte balance either by opening up other non-CFTR dependent chloride anion channels or by inhibiting cellular uptake of sodium cations. Unfortunately, the viability of this latter electrolyte balance restoration approach still remains limited.
Gene replacement therapy approaches have been successful in treating genetic diseases including adenosine deaminase deficiency, and gene therapy holds great promise in providing a cure for a variety of other genetic diseases, including cystic fibrosis. Transfection of a wide variety of cells, including human pancreatic adenocarcinoma (CFPAC) cells with the cDNA encoding normal CFTR increases transport of chloride exhibited by those cells. These studies suggest that transfection of human lung epithelia with CFTR cDNA might lead to a treatment for cystic fibrosis. Indeed, a number of past and current studies to evaluate the safety and efficacy of transfer of the CFTR to the respiratory epithelia of human cystic fibrosis patients using adenoviral and other transfer vectors have appeared or are in progress. However, such approaches may be expensive and may have associated side effects such as inflammation which must be overcome before gene therapy can become an effective routine treatment regimen for cystic fibrosis.
The activation of the defective and/or alternative functioning chloride channels in cystic fibrosis epithelial cells in order to normalize their permeability to chloride is one of the primary therapeutic goals of the treatment of cystic fibrosis and has not yet been accomplished (Boat, T. F., Welsh, M. J. and Beaudet, A. L., "Cystic Fibrosis" in The Metabolic Basis of Inherited Disease, pp. 2649-2680 (Striver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D. eds.) McGraw-Hill, New York (1989)). Thus, there exists an urgent need for a treatment that increases the permeability of epithelial cells to chloride and thereby can be used to treat cystic fibrosis. Such a treatment would be most beneficial if it were nontoxic and nonirritating to the epithelial cell linings, yet allowed the restoration of the proper chloride equilibrium of the cells, as well as the clearing of existing mucus. The present invention satisfies this need by providing methods and compounds which can therapeutically relieve both the cause of the manifestations of cystic fibrosis, as well as the manifestations themselves.
"ClC" Chloride Channels
ClC Chloride channels are widely distributed. The family of channels includes many types and many variants and isoforms of each type. They are widely distributed in nature and may play important, yet undiscovered roles in chloride transport. Indeed, the nature, role, and mechanisms of regulation of these channels and the processes they participate in are poorly understood. The following information provides a background information on ClC chloride channels.
The ClC chloride channels may play important, yet undiscovered roles in chloride transport in addition to the well known involvement in regulation of cellular volume, such as in the regulation of the resting potential of muscle membrane where defective channels lead to myotonia, and in kidney disease, where defective channels lead to Dent's disease.
The channel can be modeled as a central transmembrane region with an extracellular loop which contains the pH sensor, an N-terminal region which contains an inactivation region, and a C-terminal region which forms an activation region.
In normal mouse airway, the CFTR, a PKA-activated Cl-channel and the as yet unidentified Ca.sup.2+ activated Cl-channel contribute the bulk of the Cl-current carried by the cells. Approximately 25% of the total current is carried by the CFTR, the remainder is carried by a Ca.sup.2+ activated Cl-channels. Thus, the Ca.sup.2+ activated Cl-channel is a major contributor to Cl-transport in the airway of the mouse, and is likely responsible for he mild disease phenotype in the CFTR (-/-) mouse lung. The molecular nature of this channel is not known, despite recent advances in determination of the primary sequence of a variety of other mammalian Cl-channels. In the case of genetic mutation of this protein in human cystic fibrosis patients, Cl-transport is defective in several organ systems including the lung, gut, and pancreas, and the defect is associated with disease.
CaMKII-activated Cl-channels.
Ca.sup.2+ -activation of chloride channels in SV40 transformed cell lines from normal and CF airway epithelia is mediated by CaMKII. However, the molecular nature of this channel is unknown. Our findings have shown that ClC-2G chloride channels are activated by calmodulin kinase II ("CaMKII"). In addition, a chloride channel protein purified from bovine trachea has also been shown to be CaMKII-activated. Using antibodies raised to be bovine tracheal channel protein, a preliminary report suggested that a novel chloride channel (CaMKII-activated) had been cloned and that its structure was dissimilar to ClC channels and CFTR. However, full publication of structure of this channel has not yet appeared.
Framework for interpretation of Ca.sup.2+ -activation effects.
Ca.sup.2+ clearly activates chloride channel activity in airway epithelium. To our knowledge, the mechanisms of Ca.sup.2+ activation have not been examined in detail. Thus, increase in short-circuit current (SCC) attributable to increased chloride channel function with Ca.sup.2+ in airway epithelia may equally be attributable to increased recruitment and increased probability opening of Cl channels, mechanisms which have not been addressed in detail in the literature, largely due to difficulties of dissociation of these two types of activation mechanisms unless studies of Po of the Ca.sup.2+ -activated chloride channels are carried out in single channel studies (which have not appeared in the literature).
U.S. Pat. No. 5,242,947, Cherksey et al., issued Sep. 7, 1993, discloses methods for regulating cation transport across cellular membranes possessing cation channels. The cell membrane possessing a specific ion channel is exposed to a non-aromatic polyamine compound having a lysine- or arginine-based moiety (or a guanidine moiety) coupled to a straight chain polyamine.
U.S. Pat. No. 4,937,270, Hamilton et al., describes a method for making a water-insoluble biocompatible gel by activating HA with a carbondiimide then reacting the activated HA with a nucleophile (e.g., an amine).
U.S. Pat. No. 5,503,989, Bibbs et al., issued Apr. 2, 1996, discloses methods of preparing a peptide having a C-terminal amide group ("peptide amide") from the corresponding peptide having a C-terminal carboxyl group ("peptide acid"). Thus, a solution of peptide acid is treated with a carboxyl activating agent to give a reactive intermediate. Suitable carboxyl activating agents include carbodiimide compounds. The reactive intermediate is then treated with a trapping agent and an amine source (which is the donor --NH2) to give the peptide amide. According to a second, preferred, aspect of the invention, the peptide acid is treated with an alcohol in the presence of an acid to give a reactive intermediate different from that referred to above.
U.S. Pat. No. 5,504,241, Pohl et al., issued Apr. 2, 1996, discloses carbodiimides and/or oligomeric polycarbodiimides based on 1,3-bis(1-methyl-1-isocyanathoethyl)benzene, their preparation, and their use as hydrolysis stabilizers.
The carbodiimides have been used as stabilizers against hydrolytic cleavage of polyester-based plastics. See, e.g., U.S. Pat. No. 3,193,523.
U.S. Pat. No. 5,399,346, Anderson, issued Mar. 21, 1995, discloses the use of primary human cells which are genetically engineered with DNA (RNA) encoding a marker or therapeutic which is expressed to be expressed in vivo. Such engineered cells may be used in gene therapy.
U.S. Pat. No. 5,240,846, Collins et al, issued Aug. 31, 1993, discloses gene therapy for treating cystic fibrosis(CF). Delivery and expression of a single copy of a normal CFTR gene leads to stable correction of the Cl channel regulation defect present in CF epithelial cells. The present invention includes recombinant viral and plasmid vectors, alternative CFTR gene delivery strategies, and transduced CF cells and cell lines carrying a recombinant gene for functional CFTR. CF epithelial complementation through transduction of the present invention also provides an assay for determining the validity of other putative CF mutations. None of these references individually or collectively teach or suggest the present invention.